The study reports the cryogenic electron microscopy (cryo-EM) structures of the human adenosine A3 receptor (A3AR) bound to selective agonists CF101 and CF102, along with the heterotrimeric G1 protein, at 3.3-3.2 Å resolution. These structures reveal that the agonists reside in the orthosteric pocket, forming conserved interactions via their adenine moieties, while their 3-iodobenzyl groups exhibit distinct orientations. Functional assays highlight the critical role of extracellular loop 3 (ECL3) in A3AR's ligand selectivity and receptor activation. Key mutations, including His3.37, Ser5.42, and Ser6.52, in a unique sub-pocket of A3AR significantly impact receptor activation. Comparative analysis with the inactive A2AR structure highlights a conserved receptor activation mechanism. The findings provide comprehensive insights into the molecular recognition and signaling of A3AR, paving the way for designing subtype-selective adenosine receptor ligands.The study reports the cryogenic electron microscopy (cryo-EM) structures of the human adenosine A3 receptor (A3AR) bound to selective agonists CF101 and CF102, along with the heterotrimeric G1 protein, at 3.3-3.2 Å resolution. These structures reveal that the agonists reside in the orthosteric pocket, forming conserved interactions via their adenine moieties, while their 3-iodobenzyl groups exhibit distinct orientations. Functional assays highlight the critical role of extracellular loop 3 (ECL3) in A3AR's ligand selectivity and receptor activation. Key mutations, including His3.37, Ser5.42, and Ser6.52, in a unique sub-pocket of A3AR significantly impact receptor activation. Comparative analysis with the inactive A2AR structure highlights a conserved receptor activation mechanism. The findings provide comprehensive insights into the molecular recognition and signaling of A3AR, paving the way for designing subtype-selective adenosine receptor ligands.